Insertion Specificity of the hATx-6 Transposase of Hydra magnipapillata

Transposable elements (TE) are mobile genetic elements, present in all domains of life. They commonly encode a single transposase enzyme, that performs the excision and reintegration reactions, and these enzymes have been used in mutagenesis and creation of next-generation sequencing libraries. All transposases have some bias in the DNA sequence they bind to when reintegrating the TE DNA. We sought to identify a transposase that showed minimal sequence bias and could be produced recombinantly, using information from the literature and a novel bioinformatic analysis, resulting in the selection of the hATx-6 transposase from Hydra vulgaris (aka Hydra magnipapillata) for further study. This transposase was tested and shown to be active both in vitro and in vivo, and we were able to demonstrate very low sequence bias in its integration preference. This transposase could be an excellent candidate for use in biotechnology, such as the creation of next-generation sequencing libraries.

Structural and functional analysis of Hydra Actinoporin-Like Toxin 1 (HALT-1)

Actinoporins are a family of α-pore-forming toxins (α-PFTs) that have been identified in sea anemones. Recently, a freshwater Hydra Actinoporin-Like Toxin (HALT) gene family was found in Hydra magnipapillata. Unlike sea anemone actinoporins that use sphingomyelin as their main recognition target, the HALTs proteins may recognise alternative lipid molecules as their target. To unveil the structural insights into lipid preference of HALTs protein as compared to sea anemone actinoporins, we have determined the first crystal structure of actinoporin-like toxin, HALT-1 at 1.43 Å resolution with an acetylated lysine residue K76. Despite the overall structure of HALT-1 sharing a high structural similarity to sea anemone actinoporins, the atomic resolution structure revealed several unique structural features of HALT-1 that may influence the lipid preference and oligomerisation interface. The HALT-1 contains a RAG motif in place of the highly conserved RGD motif found in sea anemone actinoporins. The RAG motif contributed to a sharper β9-β10 turn, which may sway its oligomerisation interface in comparison to sea anemone actinoporins. In the lipid-binding region, the HALT-1 contains a shorter α2 helix and a longer α2-β9 loop due to deletion and subsequently an insertion of five amino acid residues in comparison to the sea anemone actinoporins. Structure comparison and molecular docking analysis further revealed that the HALT-1 lipid-binding site may favour sphingolipids with sulfate or phosphate head group more than the sphingomyelin. The structure of HALT-1 reported here provides a new insight for a better understanding of the evolution and lipid recognition mechanism of actinoporin.

A Reference Genome from the Symbiotic Hydrozoan, Hydra viridissima

Various Hydra species have been employed as model organisms since the 18th century. Introduction of transgenic and knock-down technologies made them ideal experimental systems for studying cellular and molecular mechanisms involved in regeneration, body-axis formation, senescence, symbiosis, and holobiosis. In order to provide an important reference for genetic studies, the Hydra magnipapillata genome (species name has been changed to H. vulgaris) was sequenced a decade ago (Chapman et al., 2010) and the updated genome assembly, Hydra 2.0, was made available by the National Human Genome Research Institute in 2017. While H. vulgaris belongs to the non-symbiotic brown hydra lineage, the green hydra, Hydra viridissima, harbors algal symbionts and belongs to an early diverging clade that separated from the common ancestor of brown and green hydra lineages at least 100 million years ago (Schwentner and Bosch 2015; Khalturin et al., 2019). While interspecific interactions between H. viridissima and endosymbiotic unicellular green algae of the genus Chlorella have been a subject of interest for decades, genomic information about green hydras was nonexistent. Here we report a draft 280-Mbp genome assembly for Hydra viridissima strain A99, with a scaffold N50 of 1.1 Mbp. The H. viridissima genome contains an estimated 21,476 protein-coding genes. Comparative analysis of Pfam domains and orthologous proteins highlights characteristic features of H. viridissima, such as diversification of innate immunity genes that are important for host-symbiont interactions. Thus, the H. viridissima assembly provides an important hydrozoan genome reference that will facilitate symbiosis research and better comparisons of metazoan genome architectures.

Reduction in Toxicity of Nano-Ag-Polyvinyl-pyrrolidone Using Hydra Proteins and Peptides during Zebrafish Embryogenesis

Hydra magnipapillata cells reduce the toxicity of silver nanomaterials to zebrafish (Danio rerio) embryos. In this study, we investigated whether Hydra protein (HP) and Hydra basal disc peptide (Hym176) materials reduce nano-Ag-polyvinylpyrrolidone (N-Ag-PVP) toxicity during embryogenesis of the nanosensitive organism zebrafish. Protein (HP) was extracted from Hydra, and peptide (Hym176) was extracted from the hydra basal disc, which is attractive to nanomaterials and related to the immune system. The experimental conditions were exposure to N-Ag-PVP, HP, N-Ag-PVP+HP, Hym176, or N-Ag-PVP+Hym176 during embryo development. N-Ag-PVP+HP group showed lower toxicity than N-Ag-PVP group. In addition, in the N-Ag-PVP+HP group formed aggregated nanomaterials (≥200 nm size) through electrostatic bonding. In the gene expression profile, HP group differed in gene expression profile compared the other experimental groups and it was no genetic toxicity. HP showed a tendency to reduce side effects and abnormal gene expression produced by N-Ag-PVP with no evidence of inherent toxicity. Considering the potential nanotoxicity effects of released nanomaterials on the ecosystem, the reduction of nanotoxicity observed with HP natural materials should be regarded with great interest in terms of the overall health of the ecosystem.

Cytotoxic and apoptosis-inducing effects of wildtype and mutated Hydra actinoporin-like toxin 1 (HALT-1) on various cancer cell lines

Background Hydra actinoporin like toxin -1 (HALT-1), is a small 18.5 kDa pore forming toxin derived from Hydra magnipapillata which has been shown to elicit strong haemolytic and cytolytic activity when in contact with cell membranes. Due to its cytotoxic potency, HALT-1 was further investigated for its potential as a toxin moiety candidate in immunotoxin developmental efforts, ideally as a form of targeted therapy against cancer. Methods In this study, wtHALT-1 (wild type) and its Y110A mutated binding domain counterpart (mHALT-1) were produced and evaluated for their cytotoxic and apoptotic effects on various cancer cell lines. A total of seven different tumour and non-tumour cell lines including HeLa, HepG2, SW-620, MCF-7, CCD841CoN, NHDF and HCT116 were used. Immunofluorescence assays were used to observe membrane binding and localization changes between both HALT-1 recombinant proteins based on 6xHis-tag detection. Result Based on MTT data, mHALT-1 demonstrated a significant reduction of 82% ± 12.21% in cytotoxic activity across all cell lines after the membrane recognition domain had been mutated in comparison to the wtHALT-1. Annexin V FITC/PI assay data also indicated that HeLa, HepG2 and MCF-7 demonstrated an apoptosis-mediated cell death after being treated with wtHALT-1. Additionally, a notable difference between wtHALT-1 and mHALT-1 binding affinity was clearly observed where emission of green fluorescence along the cell membrane was observed only in wtHALT-1 treated cells. Discussion These results suggest that mHALT-1 (Y110A) can be potentially developed as a toxin-moiety candidate for the development of future immunotoxins against various human cell-based diseases.